The long-term goal of this project is to develop ways to interfere with HIV replication and transmission, which would have major implications for preventing the spread of HIV/AIDS. Any successful preventive strategy must work before infection becomes established, and the hallmark of an established retroviral infection is integration. The viral integrase enzyme catalyzes at least two endonuclease reactions in vivo: specific nicking to prepare the ends of viral DNA for integration and nonspecific insertion of the viral DNA into cellular DNA. Integrase also has a potent nonspecific endonuclease activity that can nick any DNA sequence in vitro, and this activity is dramatically stimulated by certain small compounds. These facts suggest a novel (and ironic) antiviral strategy in which integrase is stimulated to destroy viral DNA before integration (with any damage to cellular DNA being limited to newly infected cells and also blocking infection). Thus, the objectives of this proposal are to identify potent stimulators of the nonspecific nicking activity of HIV-1 integrase and to bring at least one of these agents to the verge of clinical testing. The central hypothesis, based on known precedents and preliminary data, is that integrase's nonspecific endonuclease activity can be stimulated for a new antiviral strategy that can be part of a safe and effective combination microbicide regimen. In the R21 phase, Aim 1 will optimize a high-throughput assay for integrase-mediated nonspecific DNA nicking;Aim 2 will screen 50,000 chemicals in the Penn State Drug Development and Discovery Core for additional agents that stimulate HIV-1 integrase to nick DNA nonspecifically (with appropriate secondary assays to validate positive hits);and Aim 3 will use quantitative antiviral and cell toxicity studies to prioritize integrase stimulator (IS) compounds based on their therapeutic indices, all the while feeding back to organic chemists who will design and synthesize rational analogues of lead compounds for testing in an iterative fashion. In the R33 phase, Aim 4 will test the safety of each candidate IS compound in expanded toxicity studies, including a mouse model of cervicovaginal toxicity;and Aim 5 will test the range of antiviral activity against different subtypes of HIV-1 and in a NOD-SCID-hu mouse model of HIV-1 infection. These data will also feed back to the discovery pathway to make new derivatives of IS compounds. Finally, because the ideal microbicide regimen would combine agents that work outside cells to impede virus entry with agents that work inside cells to abort infection for any viruses that do gain entry, Aim 6 will evaluate microbicide combinations that include IS compounds for cooperative activity against HIV-1, in preparation for moving at least one drug candidate into future clinical trials.